Abstract
In order to enhance recombinant protein productivity in animal cells, we developed the oncogene activated production (OAP) system. The OAP system is based on the premise that oncogenes are able to enhance promoter activity. To this end, we constructed reported plasmids by fusing various promoters to the human interleukin-6 (hIL-6) cDNA, and the effector plasmids by inserting individual oncogenes, for example c-myc, c-fos, v-jun, v-myb and c-Ha-ras, downstream from the human cytomegalovirus immediate early (CMV) promoter. Results of transient expression experiments with BHK-21 cells suggest that the CMV promoter is the most potent promoter examined and that theras product is able to transactivate the β-actin, CMV and SRα promoters. Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with theras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify theras oncogene. We were able to rapidly establish a stable and highly productive clone which exhibited a 35-times higher production rate as compared to the control value.
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References
Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdort HJ, Jonat C, Herrich P and Karin M (1987) Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulatedtrans-acting factor. Cell 49: 729–739.
Baldari CT, Macchia G, Massone A and Telford JL (1992a) p21ras contribute to HIV-1 activation in T-cell. FEBS Lett. 304: 261–264.
Baldari CT, Macchia G and Telford JL (1992b) Interleukin-2 promoter activation in T-cells expressing activated Ha-ras *. J. Biol. Chem. 267: 4289–4291.
Barbacid M (1987)Ras genes. Annu. Rev. Biochem. 56: 779–827.
Behr JP, Demeneix B, Loeffler JP and Perez-Mutul J (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. USA 96: 6982–6986.
Bergmann DG, Souza LM and Baluda MA (1981) Vertebrate DNAs contain nucleotide sequences related to the transforming gene of avian myeloblastosis virus. J. Virol. 40: 450–455.
Chiu R, Boyle WJ, Meek J, Smeal T, Hunter T and Karin M (1988) The c-fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive gene. Cell 54: 541–552.
Cockett MI, Bebbington CR and Yarranton GT (1990) High level expression heterologous genes in animal cell using viral regulatory systems. Eur. Pat. Appl. 900718, 21.
Dasgupta P, Saikumar P, Reddy CD and Reddy EP (1990) Myb protein binds to human immunodeficiency virus 1 long terminal repeat (LTR) sequences and transactivates LTR-mediated transcription. Proc. Natl. Acad. Sci. USA 87: 8090–8094.
Friedman JS, Cofer CL, Anderson CL, Kushner JA, Gray PP, Chapman GE, Stuart MC, Lazarus L, Shine J and Kushner PJ (1989) High expression in mammalian cells without amplification. Bio/Technology 7: 359–362.
Geiser AG, Kim S-J, Roberts AB and Sporn MB (1991) Characterization of the mouse transforming growth factor-β1 promoter and activation by the Ha-ras oncogene. Mol. Cell. Biol. 11: 84–92.
Griffiths B (1990) Animal cell — the breakthrough to a dominant technology. Cytotechnology 3: 109–116.
Hayward BE, Hussain A, Wilson RH, Lyons A, Woodcook V, McIntosh B and Harris TJR (1986) The cloning and nucleotide sequence of cDNA for an amplified glutamine synthetase gene from Chinese hamster. Nucl. Acids Res. 14: 999–1008.
Ishiyama M, Shiga M, Sasamoto K, Mizoguchi M and He P (1993) A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem. Pharm. Bull. 41: 1118–1122.
Izumi M, Miyazawa H, Kamakura T, Yamaguchi I, Endo T and Hanaoka F (1991) Blasticidin S-resistance gene (bsr): A novel selectable marker for mammalian cells. Exp. Cell Res. 197: 229–233.
Kaddurah-Daouk R, Greene JM, Baldwin AS Jr, Kingston RE (1987) Activation and repression of mammalian gene expression by the c-myc protein. Genes Dev. 1: 347–357.
Kelekar A, Cole MD (1987) Immortalization by c-myc, H-ras and E1A oncogenes induce differential cellular gene expression and growth factor responses. Mol. Cell. Biol. 7: 3899–3907.
Kingston RE, Baldwin AS Jr and Sharp PA (1984) Regulation of heat shock protein 70 gene expression by c-myc. Nature 312: 280–282.
Le HV, Ramanathan L, Labdon JE, Mays-Ihinco CA, Syto R, Arai N, Hoy P, Takebe Y, Nagabhushan TL and Trotta PP (1988) Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells. J. Biol. Chem. 263: 10817–10823.
Ledwith BJ, Manam S, Kraynak AR, Nichols WW and Bradley MO (1990) Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene. Mol. Cell. Biol. 10: 1545–1555.
Lee W, Haslinger A, Karin M and Tjian R (1987) Activation of transcription by two factors that bind promoter and enhancer sequence of the human metallothionein gene and SV40. Nature 325: 368–372.
Maki Y, Bos TJ, Davis C, Starbuck M and Vogt PK (1987) Avian sarcoma virus 17 carries thejun oncogene. Proc. Natl. Acad. Sci. USA 84: 2848–2852.
Nishina Y, Nakagoshi H, Imamoto F, Gonda TJ and Ishii S (1989) Transactivation by the c-myb proto-oncogene. Nucl. Acids Res. 17: 107–117.
Sakura H, Kanei-Ishii C, Nagase T, Nakagoshi H, Gonda TJ and Ishii S (1989) Delineation of three functional domains of the transcriptional activator encode by the c-myb protooncogene. Proc. Natl. Acad. Sci. USA 86: 5758–5762.
Sambucetti LC, Cherrington JM, Wilkinson GWG and Mocarski ES (1989) NF-κB activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T-cell stimulation. EMBO J, 8: 4251–4258.
Satake M, Ibaraki T and Ito Y (1988) Modulation of polyomavirus enhancer binding protein by Ha-ras oncogene. Oncogene 3: 69–78.
Schimke RT (1984) Gene amplification in cultured animal cells. Cell 37: 705–713.
Sekiya T, Fushimi M, Hori H, Hirohashi S, Nishimura S and Sugimura T (1984) Molecular cloning and the total nucleotide sequence of the human c-Ha-ras-1 gene activated in a melanoma from a Japanese patient. Proc. Natl. Acad. Sci. USA 81: 4771–4775.
Shen X-Z, Jiang F-Y, Zhang Y-G, Li J-M and Yang W-M (1989) Expression of recombinant human growth hormone gene in mammalian cells: Effects of dual genetic elements. In: Murakami H (ed.) Trends in Animal Cell Culture Technology, pp. 271–276, Kodansha-VCH.
Shirahata S, Teruya K, Mori T, Seki K, Ohashi H, Tachibana H and Murakami H (1991) Genetic enhancement of protein productivity of animals cells by oncogenes. In: Sasaki R and Ikura K (eds.) Animal cell Culture and Production of Biologicals, pp. 259–266, Kluwer Academic Publishers.
Shirahata S, Teruya K, Seki K, Mori T, Ohashi H, Tachibana H and Murakami H (1992) Oncogene-activated production of recombinant proteins for animal cells. In: Spier RE, Griffiths JB and MacDonald (eds.) Animal cell technology: Developments, Processes and Products, pp. 54–59, Butterworth-Heinemann, Oxford.
Stacey DW, Watson T, Kung H-F and Curran T (1987) Microinection of transformingras protein induces c-fos expression. Mol. Cell. Biol. 7: 523–527.
Takai Y, Kikkawa U, Kaibuchi K and Nishizuka Y (1984) Membrane phospholipid metabolism and signal ransduction for protein phosphorylation. In: Greengard P and Robinson GA (eds.) Advances in cyclic nucleotide and protein phosphorylation research, Vol. 18: pp. 119–158, Raven Press, New York.
Takebe Y, Seiki M, Fujisawa J, Hoy P, Yokota K, Arai K, Yoshida M and Arai N (1988) SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5′ segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8: 466–472.
Taya Y, Hosogai K, Hirohashi S, Shimosato Y, Tsuchiya R, Tsuchida N, Fushimi M, Sekiya T and Nishimura S (1984) A novel combination of K-ras andmyc amplification accompanied by point mutational activation of K-ras in a human lung cancer. EMBO J. 3: 2943–2946.
Teruya, K., Shirahata S, Yano T, Seki K, Tachibana H, Ohashi H and Murakami H (1993) Secretory cell immunoscreening assay — A highly sensitive screening method for secretory cells. Anal. Biochem. 214: 468–473.
van Straaten F, Müller R, Curran T, Van Beveren C and Verma IM (1983) Complete nucleotide sequence of a human c-onc gene: Deduced amino acid sequence of the human c-fos protein. proc. Natl. Acad. Sci. USA 80: 3183–3187.
Weston K and Bishop JM (1989) Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb. Cell 58: 85–93.
Wong A, Lu S-D, and Mark DF (1984) Site-Specific mutagenesis of the human interleukin-2 gene: Structure-function analysis of the cysteine residues. Science 224: 1431–1433.
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Yano, T., Teruya, K., Shirahata, S. et al. Ras oncogene enhances the production of a recombinant protein regulated by the cytomegalovirus promoter in BHK-21 cells. Cytotechnology 16, 167–178 (1994). https://doi.org/10.1007/BF00749904
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DOI: https://doi.org/10.1007/BF00749904